PROJECT SUMMARY Quorum sensing is a form of cell-cell communication that allows members of bacterial populations to coordinate cooperative activities in a cell density-dependent fashion. Quorum sensing has been shown to play a significant role in the virulence of Pseudomonas aeruginosa and other pathogens, and P. aeruginosa has become a model for studies of basic biological principles of quorum sensing, communication, and cooperation in general. Our research on P. aeruginosa acyl-homoserine lactone quorum sensing is fundamental to efforts aimed at manipulating quorum sensing during infections, in complex microbial communities, and for biotechnological applications. We have been, and will continue to be, interested in basic mechanisms of quorum sensing, the selective pressures favoring quorum-sensing control of gene expression, and the costs and benefits of quorum sensing in P. aeruginosa. Quorum sensing functions to control and coordinate cooperative behaviors. It is clear that cooperativity is an evolved biological phenomenon, but there is considerable controversy about the selective forces allowing stable cooperativity. What are the costs and benefits of cooperativity, and what are the possible advantages to controlling cooperativity by quorum sensing? In the past five years we have made enormous advances in understanding at a molecular level how cooperation is stabilized in P. aeruginosa and how we can destabilize it. These advances are important to the field of population biology and to our understanding of the roles P. aeruginosa quorum sensing plays in certain infections. Our continued research will use molecular genetic approaches to investigate the quorum sensing control of gene expression, and we will continue to introduce new techniques and concepts into what has become a vibrant field of microbiological research. Specifically we will ask questions about the determinants of signal specificity and the potential for bacterial species to eavesdrop on each other. We will attempt to define the determinants of signal generation and reception selectivity. We want to better understand the role of quorum sensing during human infections with our initial emphasis on the chronic lung infections that plague people with the genetic disease cystic fibrosis. We have recently learned that one of the least understood components of P. aeruginosa quorum sensing circuitry, RhlR is a key quorum-sensing element in P. aeruginosa. Our recent work has allowed us to overcome the major obstacle in learning about RhlR, an inability to study it in vitro. The long term vision of this work is to understand how and why quorum sensing circuits are often layered, to understand how the diversity of quorum sensing systems has evolved to its current state, and to attain a conceptual framework to explain how communication and cooperative activities are wired to provide stability of bacterial group activities and how we might be able to use the knowledge to manipulate bacterial populations.